For laboratory use. Not for use in veterinary diagnostic procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a veterinary disease.

Co-isolation of viral RNA and bacterial DNA.

Consistent amounts of BVDV particles and Salmonella cells were simultaneously spiked into a variety of animal specimens. Samples were processed in parallel on the QIAcube HT with the cador Pathogen 96 QIAcube HT Kit or manually with the QIAamp cador Pathogen Mini Kit. Co-isolated viral RNA and bacterial DNA were identified using an in-house assay. Mean CT values are shown for [A] BVDV and [B]Salmonella. Small error bars (±1 SD of 3 extraction replicates) indicate high precision for both purification procedures.

Highly repeatable viral RNA isolation.

Blood samples of various species were spiked with BVDV particles and were frozen at –20°C. Of each sample, 4 replicates were processed on QIAcube HT with the cador Pathogen 96 QIAcube HT Kit in 3 subsequent runs on different days. [A] Viral RNA was identified using an in-house assay, which included [B] an internal amplification control. Error bars represent ±1 SD of 3 replicates. The BVDV CT values for each set of quadruplicates showed a mean relative standard deviation of 0.67 ± 0.43%, and the inter-assay CV for each sample from 3 runs ranged from 0.14 to 0.89% (mean: 0.45%), indicating high repeatability of viral RNA purification on QIAcube HT.

High performance protocol.

Consistent amounts of BVDV particles and Salmonella cells were simultaneously spiked into a variety of animal specimens. Samples were processed in parallel on the QIAcube HT with the cador Pathogen 96 QIAcube HT Kit or a kit from supplier A. Co-isolated viral RNA and bacterial DNA were identified using in-house assays. Mean CT values are shown for [A] BVDV and [B]Salmonella. Error bars represent ±1 SD of 3 extraction replicates. Samples purified on the QIAcube HT resulted in lower CT values for both bacterial DNA and viral RNA from all starting materials tested.

Purification of nucleic acids from undiluted animal whole blood is quick and simple, with minimal risk of silica membrane clogging. Automated processing of samples in 96-well format is precise and repeatable between different purification runs performed on different days, providing not only convenience but also peace of mind (see figure Highly repeatable viral RNA isolation).

The QIAcube HT platform with the cador Pathogen QIAcube HT Kit allows parallel purification of different nucleic acids from a range of sample types using just one universal instrument protocol. Co-purification of viral RNA and bacterial DNA is simple and reliable and provides the same high-quality nucleic acids as manual processing with the QIAamp cador Pathogen Mini Kit (see figure Co-isolation of viral RNA and bacterial DNA). Compared with a kit from another supplier, the cador Pathogen 96 QIAcube HT Kit displays high performance, and succeeds even when the other kit falls short (see figure High performance protocol).

Principle

The cador Pathogen 96 QIAcube HT Kit enables automated purification of pathogen nucleic acids — viral RNA and DNA and bacterial DNA — from up to 200 μl of animal whole blood, serum, plasma, swabs, or washes using the QIAcube HT instrument. By applying the pretreatments described in the cador Pathogen 96 QIAcube HT Handbook, tissue and fecal samples can also be processed. The procedure yields high-quality DNA and RNA that perform well in PCR, RT-PCR, and other downstream applications.

The cador Pathogen 96 QIAcube HT Kit combines the selective binding properties of a silica-based membrane with a high-throughput 96-well format, and is designed for fully automated, simultaneous processing of 24–96 samples on the QIAcube HT instrument.

Kit specifications

Specification

Description

Number of samples

24–96 samples (to be processed in increments of 8)

Sample input volume

Up to 200 μl

Elution volume

150 μl

Duration

96 samples in approximately 90 minutes*
24 samples in approximately 60 minutes*
Each additional 8 samples extend the processing time by 5 minutes

* Time for the standard protocol, excluding any sample pretreatments.

Procedure

Samples are lysed under highly denaturing conditions at room temperature in the presence of proteinase K and Buffer VXL, which together ensure the inactivation of nucleases. Adding Buffer ACB adjusts the binding conditions for the co-purification of DNA and RNA. The lysate is then transferred to a QIAamp 96 plate. As vacuum is applied, DNA selectively binds to the QIAamp membrane as contaminants pass through. Three efficient wash steps remove the remaining contaminants and enzyme inhibitors, and nucleic acids are eluted in Buffer AVE.

Most common fluid samples can be processed directly using the main protocol, which involves the lysis, binding, wash, and elution steps. However, depending on the starting material and the target pathogen, one of the pretreatment protocols may be needed (see the table "Pretreatments for the cador Pathogen 96 QIAcube HT Kit protocol"). After pretreatment, the samples undergo the same procedure as for fluid samples, enabling automated parallel processing on the QIAcube HT.

Pretreatments for the cador Pathogen 96 QIAcube HT Kit protocol

Name

Application

Pretreatment B1

For difficult-to-lyse bacteria* in whole blood or pretreated tissue

Pretreatment B2

For difficult-to-lyse bacteria* in cell-free fluids

Pretreatment T1

Mechanical disruption of tissue for extraction of viral RNA and DNA

Pretreatment T2

Enzymatic digestion of tissue for extraction of viral and bacterial DNA

Pretreatment F1

Isolation of viral RNA and DNA from fecal samples

Pretreatment F2

Isolation of bacterial and viral DNA from fecal samples

* Gram-positive bacteria are difficult to lyse due to their rigid cell wall. However, some Gram-negative bacteria are also difficult to lyse, and benefit from pretreatment B1 or B2.

Q Protocol notes for purification of pathogen nucleic acids from fluid samples on the QIAcube HT using the cador Pathogen 96 QIAcube HT Kit; use with the cador Pathogen 96 QIAcube HT V3.qsp run file. For use with QIAcube HT Operating Software 4.17; not for use with QIAcube HT Prep Manager Software.

These Q Protocols and installation files can only be used with QIAcube HT Operating software version 4.17 and are not compatible with the new QIAcube HT Prep Manager software.

QIAcube HT Prep Manager Software already includes all standard protocols. Additional protocols and kit configurations can be easily installed. Check for available updates under the "Operating Software" section of the "Product Resources" tab.